Conjugates of sodium channel blockers and methods of using the same

ABSTRACT

Compounds of the general formula P 1 -L-P 2 ; wherein “P 1 ” is a pyrazinoylguanidine sodium channel blocker, “L” is a linking group, and “P 2 ” is either (i) a pyrazinoylguanidine sodium channel blocker or (ii) a P2Y 2  receptor agonist, are disclosed. Pharmaceutical formulations containing the same and methods of use thereof to hydrate mucosal surfaces such as airway mucosal surfaces are also disclosed.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.09/618,978, filed Jul. 19, 2000, now U.S. Pat. No. 6,475,509 whichclaims the benefit of U.S. Provisional Application No. 60/144,479, filedJul. 19, 1999, the disclosure of which is incorporated herein byreference in its entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with Government support under Grant No. HL34322from the National Institutes of Health. The United States government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to conjugates of sodium channel blockers,and particularly covalent conjugates comprising a pyrazinoylguanidinesodium channel blocker and another compound such as anotherpyrazinoylguanidine sodium channel blocker or a P2Y₂ receptor agonist.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 4,501,729 to Boucher describes the use of respirable ornon-respirable amiloride to hydrate airway mucous secretions, and U.S.Pat. No. 5,656,256 to Boucher and Stutts describes the use of respirableor non-respirable benzamil and phenamil to hydrate lung mucussecretions. U.S. Pat. No. 5,789,391 to Jacobus describes methods oftreating sinusitis by administering uridine triphosphates (UTP) andrelated compounds such as P¹,P⁴-di(uridine-5′ tetraphosphate (U₂P₄) inorder to promote drainage of congested fluid in the sinuses.

U.S. Pat. No. 5,292,498 to Boucher describes nucleotides, particularlyP2Y₂ receptor agonists, that can be used to hydrate airway mucussecretions. Dinucleotides that can be used to hydrate airway mucussecretions are described in U.S. Pat. No. 5,635,160 to Stutts et al..Additional compounds that are P2Y₂ receptor ligands and can be used tohydrate airway mucus secretions are disclosed in W. Pendergast et al.,U.S. Pat. No. 5,837,861, along with U.S. Pat. Nos. 5,763,447 to Jacobusand Leighton, and 5,789,391 to Jacobus et al.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a compound (hereinafter alsoreferred to as an “active compound” or “active agent”) of the generalformula P₁-L-P₂; wherein “P₁” is a pyrazinoylguanidine sodium channelblocker, “L” is a linking group, and “P₂” is either (i) apyrazinoylguanidine sodium channel blocker or (ii) a P2Y₂ receptoragonist. An advantage of compounds of the present invention is that theyare substantially non-absorbable, or absorption-retardant or exhibitdelayed absorption on mucosal (e.g., airway, gastrointestinal) surfaces,thereby contributing to a prolonged mode of action and fewer systemicside effects.

A second aspect of the present invention is a composition comprising anactive compound as defined above in an effective therapeutic amount, ina pharmaceutically acceptable carrier.

A third aspect of the present invention is a method of treating amucosal surface in a subject in need thereof, comprising administeringan active compound as described herein in an amount effective to treatthe subject. In general, treatment of the subject will mean that themucosal surface being treated with a compound or composition of thepresent invention will be hydrated, or that the compound or compositionbeing used will block or otherwise retard the absorption of liquid ontoor onto the mucosal surface, or that the mucosal surface will otherwiseexhibit an increased volume of liquid on the mucosal surface.

A fourth aspect of the present invention is the use of an activecompound as described above for the preparation of a medicament fortreating a mucosal surface in a subject in need thereof, as describedherein.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth below.

BRIEF DESCRIPTION OF DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a graphical illustration of the effect of apical washout of acompound of the present invention, where wash-out correlates withcellular uptake. A range of reversibilities is shown, with the compoundCF-519 being completely reversible.

FIG. 2 is an example of a confocal microscopy assay of drug uptake intocultured airway epithelia. In this assay, a compound (10⁻⁴ M) is placedon the airway surface and fluorescence from the cells collected by x-zscanning confocal microscopy. The images shown on the left depictfluorescence in the cells 20 minutes after exposure to amiloride,benzamil and phenmnil. Quantitation of the drug uptake is graphicallyillustrated on the right in terms of units of fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, which further illustrate theinvention described herein. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

The term “alkyl” or “loweralkyl” as used herein refers to C1 to C4, C6or C8 alkyl, which may be linear or branched and saturated orunsaturated. Cycloalkyl is specified as such herein, and is typicallyC3, C4 or C5 to C6 or C8 cycloalkyl. Alkenyl or loweralkenyl as usedherein likewise refers to C1 to C4 alkenyl, and alkoxy or loweralkoxy asused herein likewise refers to C1 to C4 alkoxy. The term “aryl” as usedherein refers to C3 to C10 cyclic aromatic groups such as phenyl,naphthyl, and the like, and includes substituted aryl groups such astolyl. “Halo” as used herein refers to any halogen group, such aschloro, fluoro, bromo, or iodo. The term “hydroxyalkyl” as used hereinrefers to C1 to C4 linear or branched hydroxy-substituted alkyl, i.e.,—CH₂OH, —(CH₂)₂OH, etc. The term “aminoalkyl” as used herein refers toC1 to C4 linear or branched amino-substituted alkyl, wherein the term“amino” refers to the group NR′R″, wherein R′ and R″ are independentlyselected from H or lower alkyl as defined above, i.e., —NH₂, —NHCH₃,—N(CH₃)₂, etc. The term “oxyalkyl” as used herein refers to C1 to C4oxygen-substituted alkyl, i.e., —OCH₃, and the term “oxyaryl” as usedherein refers to C3 to C10 oxygen-substituted cyclic aromatic groups.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other animalsubjects (i.e., mammals, avians) for veterinary purposes. Mammals arepreferred, with humans being particularly preferred.

The present invention is useful in treating mucosal surfaces in asubject in need of such treatment. “Treatment” includes the hydration ofthe mucosal surface, or the blocking or retardation of the absorption ofliquid onto or into the mucosal surface, or an increase of volume ofliquid on the mucosal surface, whether by increasing water or liquid onthe mucosal surface, increasing the amount of salt on the surface, orboth. In a preferred embodiment, the mucosal surface is an airwaysurface. The term “airway surface” as used herein refers to airwaysurfaces below the larynx and in the lungs (e.g., bronchial passages,alveolar passages), as well as air passages in the head, including thesinuses and other nasal airways, and in the region above the larynx. Thepresent invention may also be used to treat mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-ureteral surfaces, ocular surfaces orsurfaces of the eye, the inner ear, and the middle ear.

Subjects that may be treated by the methods of the present inventioninclude patients afflicted with cystic fibrosis, primary ciliarydyskinesia, chronic bronchitis, chronic obstructive airway disease,artificially ventilated patients, patients with acute pneumonia, etc.Subjects that may be treated by the method of the present invention alsoinclude patients being nasally administered supplemental oxygen (whichtends to dry the airway surfaces), patients afflicted with an allergicdisease or response (e.g., an allergic response to pollen, dust, animalhair or particles, insects or insect particles, etc.) that affect nasalairway surfaces, patients afflicted with an infection caused by amicroorganism (e.g., infections caused by such organisms asStaphylococcis aureus, Haemophilus influenza, Streptococcus pneumoniae,Pseudomonas spp. etc.) of the nasal airway surfaces, an inflammatorydisease that affects nasal airway surfaces, or patients afflicted withsinusitis (wherein the active agent or agents are administered topromote drainage of congested mucous secretions in the sinuses byadministering an amount effective to promote drainage of congested fluidin the sinuses).

The compounds of the present invention can be prepared according tomethods described herein, as well as in accordance with known techniquesor variations thereof which will be apparent to skilled persons in lightof the disclosure set forth herein. See, e.g., D. Benos et al., Proc.Natl. Acad. Sci. USA 83, 8525 (1986); T. Kleyman et al., Am. J Physiol250 (Cell Physiol. 19): C165-C170 (1986); U.S. Pat. Nos. 3,313,813;4,501,729; 5,789,391; 5,292,498; 5,635,160; 5,837,861; 5,763,447; and5,789,391 (the disclosures of all patent references cited herein areincorporated by reference in their entirety).

1. Sodium Channel Blockers.

Any sodium channel blocker (i. e., P₁ or P₂ in the formula P₁-L-P₂) canbe used to carry out the present invention. Numerous pyrazinoylguanidinesodium channel blockers are disclosed in U.S. Pat. No. 3,313,813 toCragoe. Amiloride, one particular pyrazinoylguanidine sodium channelblocker, is described at Merck Index Registry No. 426 (12th Ed. 1996).Benzamil (also known as3,5-diamino-6-chloro-N-(benzylaminoaminomethylene) pyrazinecarboxamide)and phenamil (also known as3,5-diamino-6-chloro-N-(phenylaminoaminomethylene)pyrazinecarboxamide)are known compounds and are also disclosed in U.S. Pat. No. 3,313,813 toE. Cragoe.

Various additional pyrazinoylguanidine sodium channel blockers that areamiloride analogs are disclosed and described in T. Kleyman and E.Cragoe, J. Membrane Biol. 105, 1-21 (1988).

Preferred examples of active compounds that may be used to carry out thepresent invention are the pyrazinoylguanidine sodium channel blockersdisclosed in U.S. Pat. No. 3,313,813, incorporated by reference above.Such compounds have the formula:

wherein:

X is selected from the group consisting of chioro, bromo, iodo,loweralkyl, lower-cycloalkyl having from 3 to 7 carbons, phenyl,chlorophenyl, bromophenyl, Z-thio and Z-sulfonyl wherein Z is selectedfrom the group consisting of loweralkyl and phenyl-loweralkyl.Preferably, X is chloro.

Y is selected from the group consisting of hydroxyl, mercapto,loweralkyloxy, loweralkylthio, chloro, loweralkyl, lowercycloalkylhaving from 3 to 6 carbons, phenyl, amino having the structure:

wherein:

R is selected from the group consisting of hydrogen, amino, amidino,lower-cycloalkyl having 3 to 6 carbon atoms, loweralkyl,hydroxyloweralkyl, halo-loweralkyl, lower-(cycloalkylalkyl) having 3 to6 carbons in the ring, phenyl-loweralkyl, lower-(alkylaminoalkyl),lower-alkenyl, phenyl, halophenyl, and lower-alkylphenyl; alkylphenyl;

R₁ is selected from the group consisting of hydrogen, loweralkyl,loweralkenyl, and additionally;

R and R₁ can be joined to form a lower alkylene. Preferably, Y is amino.

R₂ is selected from the group consisting of hydrogen and loweralkyl.Preferably, R, R₁, and R₂ are hydrogen.

R₃ and R4 are indepenedently selected from the group consisting ofhydrogen, loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl. Preferably, R₃ is hydrogen,phenyl, or phenylalkyl. Preferably, R4 is hydrogen.

As discussed below, R₄ may be replaced with a linking group L.

2. Linking Groups

Any suitable linking group (i.e., “L” in formula P₁-L-P₂) may beemployed. The linking group may be a non-absorbable carrier moiety. Thenon-absorbable carrier moiety may be a carbohydrate, protein, peptide,polyamine, or water soluble linear polymer. Water soluble linearpolymers useful as carrier moieties include polyvinylpyrrolidone,polyethylene glycol, nonylphenol ethoxylates, and polyvinyl alcohol.Carbohydrates useful as carrier moieties include sugars andpolysaccharides, such as dextran, lactose, and mannitol. An additionalexample is agarose. Proteins or peptides useful as carrier moietiesinclude albumin (for example, human serum albumin) and protamine.Polyamnines useful for carrying out the present invention includespermine and spermidine.

The linking groups may be the same as those groups set forth for R₄above, except that they are provided in divalent rather than univalentform. Linking groups may also be heteroatoms, such as —O. Thus thelinking group may be an alkylene, alkylenecarbonyl, carbonylalkylene, ora carbonyl group, as follows:

where n is 0 (i.e., a direct covalent linkage) or is from 1 to 6. Suchalkylene groups may be saturated or unsaturated, and may be substituted1, 2, 3, or 4 times with C 1 -C4 alkyl, halo, phenyl, orhalo-substituted phenyl. Examples are as follows:

A phenyl or phenylene group, or two or more linked phenylene groups, maybe provided as the linking group, which phenylene group may optionallybe substituted 1, 2, 3 or four times with a halogen or alkyl group.Examples are as follows:

A substituted or unsubstituted phenylene group may be joined at eitheror both ends with a substituted or unsubstituted alkylene,alkylenecarbonyl, carbonylalkylene, or carbonyl group as described aboveto provide a linking group. Examples are as follows:

A substituted or unsubstituted alkylene, alkylenecarbonyl,carbonylalkylene, or carbonyl group as described above may joined ateither or both ends to a substituted or unsubstituted phenylene group asdescribed above to provide a linking group. Examples are as follows:

wherein “n” is as defined above. Such compounds may be furthersubstitued at either or both ends by a substituted or unsubstitutedalkylene, alkylenecarbonyl, carbonylalkylene, or carbonyl group, asdescribed above, to provide still further linking groups. Examples areas follows:

where “n” is as defined above.

3. P2Y₂ Receptor Agonists

As noted above P₂ may also be a P2Y₂ receptor ligand, such as anucleotide (e.g., ATP, UTP), dinucleotide (described in more detailhereinbelow), or derivative thereof. P2Y₂ receptor ligands that can beused to carry out the present invention include all of the compounds,particularly the nucleotides and dinucleotides that are P2Y2 ligands andare disclosed in W. Pendergast et al., U.S. Pat. No. 5,837,861 (Nov. 17,1998), along with all the compounds disclosed in U.S. Pat. Nos.5,763,447 to Jacobus and Leighton, 5,789,391 to Jacobus et al.,5,635,160 to Stutts et al., and 5,292,498 Boucher, the disclosures ofall of which are incorporated herein by reference in their entirety.

Examples of such nucleotides are depicted in Formulae I-IV

wherein:

X₁, X₂ and X₃ are each independently either O⁻or S⁻; preferably, X₂ andX₃ are O⁻;

R₁ is O, imido, methylene or dihalomethylene (e.g., dichloromethylene ordifluoromethylene); preferably, R₁ is oxygen or difluoromethylene;

R₂ is H or Br; preferably, R₂ is H; particularly preferred compounds ofFormula I are uridine 5′-triphosphate (UTP) and iiridine5′-0-(3-thiotriphosphate) (UTPγS).

A dinucleotide is depicted by the general Formula II:

wherein:

X is oxygen, methylene, difluoromethylene, imido;

n=0,1, or 2;

m=0,1, or 2;

n+m=0,1,2,3, or 4; and

B and B′ are each independently a purine residue or a pyrimidine residuelinked through the 9- or 1-position, respectively;

Z=OH or N₃;

Z′=OH or N₃;

Y=H or OH;

Y′=H or OH;

provided that when Z is N₃, Y is H or when Z′ is N₃, Y′ is H.

The furanose sugar is preferably in the β-configuration.

The furanose sugar is most preferably in the β-D-configuration.

Preferred compounds of Formula II are the compounds of Formula IIa:

wherein:

X=O;

n+m=1 or 2;

Z, Z′, Y, and Y′=OH;

B and B′ are defined in Formulas IIc and IId;

X=O;

n+m=3 or 4;

Z, Z′, Y, and Y′=OH;

B=uracil;

B′ is defined in Formulas IIc and IId; or

X=O;

n+m=1 or 2;

Z, Y, and Y′=OH;

Z′=H;

B=uracil;

B′ is defined in Formulas IIc and IId; or

n+m=0,1, or 2;

Z and Y=OH;

Z′=N₃;

Y′=H;

B=uracil;

B′=thymine; or

X=O;

n+m=0, 1, or 2;

Z and Z′=N₃;

Y and Y′=H;

B and B′=thymine; or

X=CH₂, CF₂, or NH;

n and m=1;

Z, Z′, Y, and Y′=OH;

B and B′are defined in Formulas IIc and IId .

Another preferred group of the compounds of Formula II are the compoundsof Formula IIb or the pharmaceutically acceptable salts thereof:

wherein:

X is oxygen, methylene, difluoromethylene, or imido;

n=0 or 1;

m=0 or 1;

n+m =0, 1, or 2; and B and B′ are each independently a purine residue,as in Formula IIc, or a pyrimidine residue, as in Formula IId, linkedthrough the 9- or 1- position, respectively. In the instance where B andB′ are uracil, attached at N-1 position to the ribosyl moiety, then thetotal of m+n may equal 3 or 4 when X is oxygen. The ribosyl moieties arein the D-configuration, as shown, but may be L-, or D- and L-. TheD-configuration is preferred.

Formula IIc

The substituted derivatives of adenine include adenine 1-oxide; 1,N⁶-(4-or 5-substituted etheno) adenine; 6-substituted adenine; or8-substituted aminoadenine, where R′ of the 6- or 8-HNR′ groups arechosen from among: arylalkyl (C₁₋₆) groups with the aryl moietyoptionally functionalized as described below; alkyl; and alkyl groupswith functional groups therein, such as:([6-aminohexyl]carbamoylmethyl)-, and ω-acylated-aminothydroxy, thioland carboxy) derivatives where the acyl group is chosen from among, butnot limited to, acetyl, trifluroroacetyl, benzoyl, substituted-benzoyl,etc., or the carboxylic moiety is present as its ester or amnidederivative, for example, the ethyl or methyl ester or its methyl, ethylor benzamido derivative. The ω-amino(hydroxy, thiol) moiety may bealkylated with a C₁₋₄ alkyl group.

Likewise, B or B′ or both in Formula IIb may be a pyrimidine with thegeneral formula of FIG. IId, linked through the 1 -position:

FIG. IId

wherein:

R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkoxy, C₁₋₆alkylamino, and dialkylamino, the alkyl groups optionally linked to forma heterocycle;

R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅ alkanoyl, benzoyl, orsulphonate;

R₆ is hydroxy, mercapto, alkoxy, aralkoxy, C₁₋₆-alkylthio, C₁₋₅disubstituted amino, triazolyl, alkylamino, or dialkylamino, where thealkyl groups are optionally linked to form a heterocycle or linked toN-3 to form an optionally substituted ring;

R₇ is hydrogen, hydroxy, cyano, nitro, alkenyl, with the alkenyl moietyoptionally linked through oxygen to form a ring optionally substitutedon the carbon adjacent to the oxygen with alkyl or aryl groups,substituted alkynyl or hydrogen where R₈ is amino or substituted aminoand halogen, alkyl, substituted alkyl, perhalomethyl (e.g., CF₃), C₂₋₆alkyl, C₂₋₃ alkenyl, or substituted ethenyl (e.g., allylamino, bromvinyland ethyl propenoate, or propenoic acid), C₂₋₃ alkynyl or substitutedalkynyl when R₆ is other than amino or substituted amino and togetherR₅-R₆ may form a 5- or 6-membered saturated-or unsaturated ring bondedthrough N or O at R₆, such a ring may contain substituents thatthemselves contain functionalities;

R₈ is hydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio,carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, orphenylthio.

In the general structure of FIG. IId above, the dotted lines in the 2-to 6—positions are intended to indicate the presence of single or doublebonds in these positions; the relative positions of the double or singlebonds being determined by whether the R₄, R₆, and R₇ substituents arecapable of keto-enol tautomerism.

In the general structures of FIG. IIc and IId above, the acyl groupsadvantageously comprise alkanoyl or aroyl groups. The alkyl groupsadvantageously contain 1 to 8 carbon atoms, particularly 1 to 4 carbonatoms optionally substituted by one or more appropriate substituents, asdescribed below. The aryl groups including the aryl moieties of suchgroups as aryloxy are preferably phenyl groups optionally substituted byone or more appropriate substituents, as described below. The abovementioned alkenyl and alkynyl groups advantageously contain 2 to 8carbon atoms, particularly 2 to 6 carbon atoms, e.g., ethenyl orethynyl, optionally substituted by one or more appropriate substituentsas described below. Appropriate substituents on the above-mentionedalkyl, alkenyl, alkynyl, and aryl groups are advantageously selectedfrom halogen, hydroxy, C₁₋₄ alkoxy, C₁₋₄ alkyl, C₆₋₁₂ arylalkoxy,carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic,amino, and substituted amino wherein the amino is singly or doublysubstituted by a C₁₋₄ alkyl, and when doubly substituted, the alkylgroups optionally being linked to form a heterocycle.

For purposes of further clarifying the foregoing descriptions ofFormulae IIc and IId, the descriptions can be simplified to thefollowing:

R₂ is O or is absent; or

R₁ and R₂ taken together may form optionally substituted 5-memberedfused imidazole ring; or

R₁ of the 6-HNR₁ group or R₃ of the 8-HNR₃ group is chosen from thegroup consisting of:

(a) arylalkyl (C₁₋₆) groups with the aryl moiety optionally substituted,

(b) alkyl,

(c) ([6-aminohexyl]carbamoylmethyl),

(d) ω-amino alkyl (C₂₋₁₀),

(e) ω-hydroxy alkyl (C₂₋₁₀),

(f) ω-thiol alkyl (C₂₋₁₀),

(g) ω-carboxy alkyl (C₂₋₁₀),

(h) the ω-acylated derivatives of (b), (c) or (d) wherein the acyl groupis either acetyl, trifluroacetyl, benzoyl, or substituted-benzoylalkyl(C₂₋₁₀), and

(i) ω-carboxy alkyl (C₂₋₁₀) as in (e) above wherein the carboxylicmoiety is an ester or an amide;

Formula IId

wherein:

R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkylthio, C₁₋₆alkoxy, C₁₋₆ alkylamino or dialkylamino, wherein the alkyl groups ofsaid dialkylarnino are optionally linked to form a heterocycle;

R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅ alkanoyl, benzoyl, orsulphonate;

R₆ is hydroxy, mercapto, alkoxy, aralkoxy, C₁₋₆-alkylthio, C₁₋₅disubstituted arnino, triazolyl, alkylamino or dialkylamino, wherein thealkyl groups of said dialkylamino are optionally linked to form aheterocycle or linked to N³ to form an optionally substituted ring;

R₅ - R₆ together forms a 5 or 6-membered saturated or unsaturated ringbonded through N or O at R₆, wherein said ring is optionallysubstituted;

R₇ is selected from the group consisting of:

(a) hydrogen,

(b) hydroxy,

(c) cyano,

(d) nitro,

(e) alkenyl, wherein the alkenyl moiety is optionally linked throughoxygen to form a ring optionally substituted with alkyl or aryl groupson the carbon adjacent to the oxygen,

(f) substituted alkynyl

(g) halogen,

(h) alkyl,

(i) substituted alkyl,

(j) perhalomethyl,

(k) C₂₋₆ alkyl,

(l) C₂₋₃ alkenyl,

(m) substituted ethenyl,

(n) C₂₋₃ alkynyl and

(o) substituted alkynyl when R₆ is other than amino or substitutedamino;

R₈ is selected from the group consisting of:

(a) hydrogen,

(b) alkoxy,

(c) arylalkoxy,

(d) alkylthio,

(e) arylalkylthio,

(f) carboxamidomethyl,

(g) carboxymethyl,

(h) methoxy,

(i) methylthio,

(j) phenoxy and

(k) phenylthio.

CTP and its analogs are depicted by general Formula III:

Formula III

wherein:

R₁, X₁, X₂ , and X₃ are defined as in Formula I;

R₅ and R₆ are H while R₇ is nothing and there is a double bond betweenN-3 and C-4 (cytosine), or

R₅, R₆ and R₇ taken together are —CH═CH—, forming a ring from N-3 to N-4with a double bond between N-4 and C-4 (3,N₄-ethenocytosine) optionallysubstituted at the 4- or 5-position of the etheno ring.

ATP and its analogs are depicted by general Formula IV:

Formula IV

wherein:

R₁, X₁, X₂, and X₃ are defined as in Formula I;

R₃ and R₄ are H while R₂ is nothing and there is a double bond betweenN-1 and C-6 (adenine), or

R₃ and R₄ are H while R₂ is O and there is a double bond between N-1 andC-6 (adenine 1-oxide), or

R₃, R_(4,) and R₂ taken together are —CH═CH—, forming a ring from N-6 toN-1 with a double bond between N-6 and C-6 (l,N6-ethenoadenine).

For simplicity, Formulas I, II, III, and IV herein illustrate the activecompounds in the naturally occurring D-configuration, but the presentinvention also encompasses compounds in the L-configuration, andmixtures of compounds in the D-and L-configurations, unless otherwisespecified. The naturally occurring D-configuration is preferred.

Some compounds of Formulas I, II, III, and IV can be made by methodswhich are well known to those skilled in the art and in accordance withknown procedures (Zamecnik, P., et al., Proc. Natl Acad. Sci. USA89:2370-2373 (1992); Ng, K., et al., Nucleic Acids Res. 15:3572-3580(1977); Jacobus, K. M., et al., U.S. Pat. No. 5,789,391 and Pendergast,W., et al., International Patent Application WO98/34942)); some arecommercially available, for example, from Sigma Chemical Company, PO Box14508, St. Louis, Mo. 63178. The synthetic methods of U.S. Pat. No.5,789,391 and International Patent Application WO98/34942 areincorporated herein by reference in their entirety.

Thus, examples of compounds that can be used to carry out the presentinvention include compounds of Formula I-IV above, and include compoundshaving the general formula:

wherein:

X may be O or S;

A is a purine or pyrimidine base (e.g., adenine, guanine, thymine,cytosine, uracil)(each purine or pyrimidine base is preferably joined tothe ribose or deoxyribose ring by covalent bond to the 9 nitrogen in thecase of purines, or by covalent bond to the 1 nitrogen in the case ofpyrimidines);

R₁ is H or OH; and

n is from 1 to 4or 6,preferably 2, 3 or 4.

Additional examples of receptor agonists that can be used to carry outthe present invention are dinucleotides, including those having thegeneral formula:

wherein:

A and B are each independently a purine or pyriaidine base (e.g.,adenine, guanine, thymine, cytosine, uracil); preferably, A is uraciland B is cytosine;

R₁ and R₂ are each independently selected from the group consisting of Hor OH; and n is from 1 to 6, preferably 3 or 4.

For P2Y₂ receptor ligands as described herein, the linking group may becovalently joined to the purine or pyrimidine base, or the correspondingribose or deoxyribose ring (e.g., of the compounds of Formula I-IVabove), or attached to the terminal phosphate moiety of compoundsrepresented by Formulae I, II and IV above, by any suitable means, suchas by covalently joining the linking group thereto in any suitableposition (e.g., a ring carbon such as the 5 carbon in a pyrirnidine, orthe 2, 6 or 8 carbon in a purine), to which linking group the ligand maybe covalently attached.

4. Example Conjugate Compounds

Specific examples of active compounds of the present invention, where P₂is a pyrazinoylguanidine sodium channel blocker, include but are notlimited to the following:

Additional examples of conjugate compounds useful in the presentinvention include those compounds whose structures are shown in Table 1,below, and in the Examples that follow.

Examples of active compounds of the present invention, where P₂ is aP2Y₂ receptor ligand, are as follows:

5. Pharmaceutically Acceptable Salts

The term “active agent” as used herein, includes the phan-naceuticallyacceptable salts of the compound, such as (but not limited to) benzamilhydrochloride or phenamil hydrochloride. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (b) salts formed fromelemental anions such as chlorine, bromine and iodine.

For nucleotides or dinucleotide active compounds, the compounds may beprepared as an alkali metal salt such as sodium or potassium, analkaline earth metal salt, or an ammonium and tetraalkyl ammonium salt,NX₄ ⁺ (wherein X is a C₁₋₄) alkyl group. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.

Active agents used to prepare compositions for the present invention mayalternatively be in the form of a pharmaceutically acceptable free baseof active agent. Because the free base of the compound is less solublethan the salt, free base compositions are employed to provide moresustained release of active agent to the lungs. Active agent present inthe lungs in particulate form which has not gone into solution is notavailable to induce a physiological response, but serves as a depot ofbioavailable drug which gradually goes into solution.

6. Formulations and Administration

A third aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g, an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibit the reabsorption of water byairway surfaces, including nasal airway surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, parenterally (e.g.,by intraveneous, intramuscular, or intraperitoneal injection), orally,rectally, via inhalation, transdermally, etc. For example, for thetreatment of constipation, the active compounds may be administeredorally or rectally to the gastrointestinal mucosal surface. The activecompound may be combined with a pharmaceutically acceptable carrier inany suitable form, such as sterile physiological saline for aninjectable or topical solution, as a droplet, tablet or the like fororal administration, as a suppository for rectal or genito-ureteraladministration, etc. Excipients may be included in the formulation toenhance the solubility of the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological saline solution or distilled water. Forexample, the active compounds may be prepared as formulations andadministered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

In one preferred embodiment they are administered by administering anaerosol suspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of the active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³,10⁻², or 10⁻¹Moles/liter, and morepreferably from about 10⁻⁶ to about 10⁻⁴ Moles/liter.

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt such as benzamil hydrochloride orphenamil hydrochloride to provide both early release of and sustainedrelease of active agent for dissolution into the mucous secretions ofthe nose. Such a composition serves to provide both early relief to thepatient, and sustained relief over time. Sustained relief, by decreasingthe number of daily administrations required, is expected to increasepatient compliance with a course of active agent treatments.

Solid or liquid particulate active agent prepared for practicing thepresent invention should as noted above include particles of respirableor non-respirable size: that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornonrespirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject of from about 10⁻⁹, 10⁻⁸, or 10⁻⁷to about 10⁻³, 10⁻², or 10⁻¹ Moles/liter, and more preferably from about10⁻⁶ to about 3×10⁻⁴ Moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.1, 0.5 or 1 to 10 or 20 milligramsof active agent particles for a human subject, depending upon the ageand condition of the subject. A currently preferred unit dose is about0.005 milligrams of active agent given at a regimen of fouradministrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating in a gelatin capsule).

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Naim, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed1995). Pharmaceutical formulations suitable for nasal administration maybe prepared as described in U.S. Pat. Nos. 4,389,393 to Schor; 5,707,644to Illum; 4,294,829 to Suzuki; and 4,835,142 to Suzuki; the disclosuresof which are incorporated by reference herein in their entirety.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. The carriermust, of course, be acceptable in the sense of being compatible with anyother ingredients in the formulation and must not be deleterious to thepatient. The carrier may be a solid or a liquid, or both, and ispreferably formulated with the compound as a unit-dose formulation, forexample, a capsule, which may contain from 0.5% to 99% by weight of theactive compound. One or more active compounds may be incorporated in theformulations of the invention, which formulations may be prepared by anyof the well-known techniques of pharmacy consisting essentially ofadmixing the components.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas sterile saline solution or sterile water. Administration may be witha pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See,e.g., U.S. Pat. Nos. 4,501,729 and 5,656,256. Suitable formulations foruse in a nasal droplet or spray bottle or in nebulizers consist of theactive ingredient in a liquid carrier, the active ingredient comprisingup to 40% w/w of the formulation, but preferably less than 20% w/w. Thecarrier is typically water (and most preferably sterile, pyrogen-freewater) or a dilute aqueous alcoholic solution, preferably made isotonicwith body fluids by the addition of, for example, sodium chloride.Optional additives include preservatives if the formulation is not madesterile, for example, methyl hydroxybenzoate, antioxidants, flavoringagents, volatile oils, buffering agents and surfactants.

Mists or aerosols of solid particles comprising the active compound maylikewise be produced with any solid particulate medicament aerosolgenerator. Aerosol generators for administering solid particulatemedicaments to a subject produce particles which are respirable ornon-respirable, as explained above, and generate a volume of mist oraerosol containing a predetermined metered dose of a medicament at arate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powderswhich may be delivered by means of an insufflator or taken into thenasal cavity in the manner of a snuff. In the insufflator, the powder(e.g., a metered dose thereof effective to carry out the treatmentsdescribed herein) is contained in capsules or cartridges, typically madeof gelatin or plastic, which are either pierced or opened in situ andthe powder delivered by air drawn through the device upon inhalation orby means of a manually-operated pump. The powder employed in theinsufflator consists either solely of the active ingredient or of apowder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically comprises from 0.1 to 100 w/w of the formulation. Asecond type of illustrative aerosol generator comprises a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquified propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume,typically from 10 to 150 or 200 μl to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol, surfactants (such as oleic acid or sorbitantrioleate), antioxidants and suitable flavoring agents.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

7. Covalent Conjugate of a Pyrazinoylguanidine Sodium Channel Blockerand a Non-absorbable Carrier Moiety

In addition to the foregoing, the pyrazinoylguanidine sodium channelblockers described above can be conjugated to a non-absorbable carriermoiety as described above to provide compounds that are active inhydrating mucosal surfaces. Some of these compounds have the formula:

wherein: X, Y, R₂ and R₃ are as defined above, and Z is a non-absorbablecarrier moiety as described above covalently linked to the adjacentnitrogen atom; or a pharmaceutically acceptable salt thereof. Suchcompounds can be prepared, formulated and administered in essentiallythe same manner as described above for the same uses as described above.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these Examples,Proton NMR spectra (360 MHz) and carbon NMR spectra (90 MHz) wereobtained on a Bruker WM-360 spectrometer using tetramethylsilane as aninternal standard. Liquid chromatography (LC)/mass spectroscopy (MS) wasperformed on a Perkin Elmer Sciex API 100 by one of the followingmethods:

Method A:YMC Pro C8 column, 5μ, 150×4.6 mm; Mobile phase A=water +0.4%acetic acid, B=acetonitrile (MeCN)+0.4% acetic acid; Gradient: 5% B for1 min, to 80% B in 7 min. followed by 100% B for 5 min.

Method B: YMC Pro C8 column, 5μ, 150×4.6 mm; Mobile Phase A=water +0.4%acetic acid, B=MeCN+0.4% acetic acid; Gradient: 5% B for 1 min, going upto 80% B in 5 min.

Method C: Luna C8 (2)column, 150×4.6 mm, 5 μ, detector λ=360 nm, mobilephase A=water+0.4% acetic acid, B=MeCN +0.4% acetic acid; Gradient: 5% Bfor 1 min, to 80% B in 7 min, followed by washout with 100% B for 5 min.

Analytical HPLC was performed on a Shimadzu HPLC 10Avp by one of thefollowing methods:

Method D: Luna C18(2) column, 5μ, 250×4.6 mm; detector λ=360 nm;Gradient: A=water +0.1% trifluoroacetic acid(TFA), B=MeCN +0.1% TEA,concentration of MeCN increases from 10 to 60% during a 0-11 mininterval, then 60-100% from 11-12 min.

Method E:Symmetry C8 column, 150×4.6 mm; detector λ=360 nm;Gradient:A=water+0.1% TFA, b=MeCN +0.1% TFA, concentration of Bincreases in the A/B mixture from 10 to 60% during the 0-11 mininterval, then B increases to 60-100% from 11-12 min.

Preparative HPLC was performed on a Gilson CombiChem by methodsdescribed in below in the Examples.

EXAMPLE 1 Synthesis of Dimeric Compounds

Referring to Scheme 1, dimer compounds of Formula I have beensynthesized as shown in TABLE 1. The synthesis begins withl-(3,5-diamino-6-chloropyrazinoyl)-2-methyl-2-thiopseudourea hydroiodide(intermediate II, prepared as described in U.S. Pat. No. 4,246,406 toCragoe et al) . . . Intermediate II was treated withN-(benzyloxycarbonyloxy)succinimide and triethylamine inN,N-dimethylformamide (DMF) to give the carbobenzyloxy(Cbz)-protectedintermediate III. Formation of the Cbz-protected dimer V occurs upontreatment of intermediate III with the appropriate diamine IV in thepresence of mercury(II) chloride and triethylamine in DMF (conditionsreported by W. Su, Synth. Comm., 26, 407-413 (1996) for the preparationof Cbz-protected guanidines). Treatment of dimer intermediate V withhydrobromic acid in acetic acid removes both of the Cbz-protectinggroups to give amiloride dimer I as the dihydrobromide salt. Thehydrobromide salts of I could be converted to the free base of I bytreatment with a strong base like potassium hydroxide in aqueous media.The free base can then be converted to other salt forms (e.g,.hydrochloride salt or other pharmaceutically acceptable salt forms) bytreatment with the appropriate acid.

TABLE 1 Dimers Max Effect of Inhibition washout (3) Compound IC50 (%)from 100% = (AMR code) Structure (nM) baseline Baseline CF-509 1363

1275 79 77 CF-510 1390

81 73 21 CF-511 1438

114 86 54 CF-512 1465

197 92 60 CF-514 1504A

121 96 20 CF-515 1504

133 99 12 CF-516 1527

330 90 65 *CF-519 1604

1075 85 100

EXAMPLE 2 THROUGH 10 Preparation of Dimeric Analogues of AmilorideEXAMPLE 2N-Cbz-1-(3,5-diamino-6-chloropyrazinoyl)1y)-2-methyl-pseudothiourea(III)

1 -(3,5 -Diamino-6-chloropyrazinoyl)-2-methyl-pseudothiourea hydroiodide(II, 494 mg, 1.27 mmol) was dissolved in a mixture of anhydrous DMF(10mL), and triethylamine (3 mL) followed by treatment withN-(benzyloxycarbonyloxy) succinimide (470 mg, 1.7 mmol) dissolved inDMF(3 mL). The reaction mixture was stirred overnight at roomtemperature. After this time, the reaction mixture was concentratedunder reduced pressure and the residue suspended in ethyl acetate (30mL). Silica gel (25 g) was added to the solution and the solvent wasevaporated to leave the silica gel impregnated with the crude productthat was purified by flash chromatography on a FlashElute™ system fromElution Solution (P.O. Box 5147, Charlottesville, Va. 22905) using a 90g silica gel cartridge (eluent:hexanes, ethyl acetate=1:2). The purifiedN-Cbz-1-(3,5-diamino-6-chloropyrazinoyl)-2-methyl-pseudothiourea (III)was yield);¹H NMR (360 MHz, DMSO-d₆) δ2.33 (s, 3H), 2.61 (s, 3H), 4.99(s, 2H), 7.39 (m, 10H), 13.7 (s, 1H); API MS m/z=395[C₁₅H₁₅ClN₆O₃S+H]³⁰; LC/MS (Method A)>99%, t_(r=)10.1 min.

EXAMPLE 31,5-Bis[(3,5-diamino-6-chloropyrazinoyl)/guanidino]-3-oxa-pentaneDihydrobromide (Ia)

A solution of 1,5-diamino-3-oxa-pentane (IVa, 30 μL, 0.3 mmol) in dryDMF (100 μL) was added to intermediate III (226 mg, 0.6 mmol) andstirred in anhydrous DMF(10 mL). Triethylamine (480 μL, 3.4 mmol) in DMF(1mL) and mercury(II) chloride (154 mg, 0.6 mmol) in DMF(100 μL) wereadded and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was filtered through silica gel andthe filtrate concentrated under reduced pressure. The residue waspurified by flash chromatography on FlashElute™ system from ElutionSolution using 90 g silica gel cartridge (eluent: ethyl acetate, hexanes=7:1). The fractions were analyzed by LC/MS (Method B) and thosefractions containing the desired product were combined and concentratedto give 1,5-bis[(N-Cbz-3,5-diamino-6-chloropyrazinoyl)guanidino]-3-oxa-pentane (Va, 102 mg, 44% yield); LC/MS>99% (Method B);API MS m/z=797 [C₃₂H₃₄Cl₂N₁₄O₇+H]⁺.

Intermediate Va (50 mg) was dissolved in 30% HBr in acetic acid (10 mL)and the mixture was stirred for 2 d. The volume of the reaction mixturewas reduced (to 4 mL) when a precipitate formed. Ethyl ether (10 mL) wasadded to the acetic acid/product mixture and the precipitate wascollected by vacuum filtration, washed with additional ether, dried andthen purified by preparative HPLC on Luna column [C18(2), 5μ, 250×21.2mm; mobile phase MeCN/water containing 0.1% TFA; gradient: 5% MeCN fromthe 0-2 min interval, increased from 5% -40% MeCN from 2-10 min, 40%-80%MeCN from 10-19 min, 40% -80% MeCN from 19-23 min, 80%-100% MeCN and100% MeCN from 23-25 min.] Fractions containing the target compound werecombined, concentrated under reduced pressure and the residueredissolved in 10% HBr and evaporated to dryness and washed with THF.The product Ia was obtained as a yellow powder: 18.9 mg (41% yield fromV); ¹H NMR (360 MHz, DMF-d₇) δ3.80 (m, 4H), 3.88 (m, 4H), 7.51 (br s,4H), 9.58 (m, 2H), 10.97 (s, 2H). API MS m/z=529 [C₁₆H₂₂Cl₂N₁₄O₃+H]³⁰;HPLC (Method D)>99%, t_(r)=6.72 min.

EXAMPLE 4 1,4-Bis [(3,5-diamino-6-chloropyrazinoyl)guanidino]butanedihydrobromide (Ib)

A solution of 1,4-diaminobutane (IVb, 24 mg, 0.3 mmol) in dry DMF (230μL) was added to III (213 mg, 0.54 mmol) in anhydrous DME(10 mL),followed by addition of triethylamine (480 μL, 3.4 mmol) in DMF (1 mL)and mercury(II), chloride (146 mg, 0.53 mmol) in DMF (600 μL). Thereaction mixture was stirred for 3 d at room temperature, then filteredthrough silica gel. The filtrate was concentrated under reduced pressureand the residue was dissolved in 30% HBr in acetic acid (20 mL) andstirred overnight at rt. The reaction mixture was poured into ethylether (150 mL) resulting in the formation of a precipitate that wasisolated by vacuum filtration and washed with water (3×0.5 mL). Thesolid precipitate was purified by preparative HPLC on a Luna C18(2)column [5 μ, 250×21.2 mm; flow rate =20 mL/min; mobile phase consists ofMeCN/water containing 0.1% TFA; gradient: 10% MeCN from the 0-2 mininterval, concentration of MeCN increased from 10% -40% from 2-10 min,40% - 100% MeCN from 10-19 min, 100% MeCN from 19-23 min, MeCN decreasedfrom 100% -10% from 23-25 min]. Fractions containing the target compoundwere combined and concentrated under reduced pressure to give a residuethat was redissolved in 10% HBr and evaporated to dryness and washedwith ethyl ether to give Ib as a pale yellow solid: 19.4 mg (10.1%yield); ¹H NMR (360 MHz, DMSO-d₆) δ1.62 (br s, 4H), 7.43 (s, 4H), 8.77(br s, 2H), 8.89 (br s, 2H), 9.24 2H), 10.48 (s, 2H); ¹³C NMR (90 MHz,DMSO-d₆) δ24.8, 40.4, 108.9, 119.5, 153.1, 154.2, 155.9 and 165.1; APIMS=513 [C₁₆H₂₂Cl₂N₁₄O₂+H]³⁰; HPLC (Method D) >99%, t_(r) 6.26 min.

EXAMPLE 5 1,5-Bis [(3,5-diamino-6-chloropyrazinoyl)guanidino]hexaneDihydrobromide (Ic)

Compound Ic was prepared following the same procedure described for Ib.The Cbz-protected pseudothiourea III (226 mg, 0.6 mmol) and1,6-diaminohexane (IVc, 34.9 mg, 0.3 mmol) reacted in the presence oftrietliylamine (480 μL, 3.4 mmol) and mercury(II) chloride (162.9 mg,0.6 mmol) to give the crude intermediate Vc, which was treated with 30%HBr in acetic acid as previously described. The crude product waspurified by preparative HPLC on a Luna C 18(2) column [5μ, 250×21.2 mm;flow rate=20 mL/min; mobile phase: MeCN/water (containing 0.1 % TFA);gradient: 15% MeCN for 0-2 min interval, increase concentration of MeCNfrom 15% -30% from 2-10 min, 30% -50% MeCN from 10-19 min, 50% -100%MeCN from 19-23 min, then decrease concentration MeCN from 100% -15%from 23-25 min]. Fractions containing the target compound were combinedand concentrated under reduced pressure to give a residue that wasredissolved in 10% HBr and evaporated to dryness and washed with ethylether to give Ic: 28.8 mg (13.5% yield based on III);¹H NMR(360 MHz,DMSO-d₆,) δ1.38 (br s, 4H), 1.59 (br s, 4H), 3.38 (m, 2H), 7.44 (s, 4H),8.75 (br s, 2H), 8.90 (br s, 2H), 9.19 (s, 2H) and 10.47 (s, ¹³C NMR (90MHz, DMSO-d₆,) δ25.5, 27.5, 40.9, 108.9, 119.6, 153.1, 154.2, 155.8 and165.1; API MS m/z=541 [C₁₈H₂₆Cl₂N₁₄O₂+H]³⁰; HPLC (Method D) 95.2%,t_(r)=7.26 min.

EXAMPLE 6 1. 3-Bis [(35-diamino-6-chloropyrazinoyl)guanidino]xylyleneDihydrobromide (Id)

Compound Id was prepared following the same procedure described for Ib.Triethylamine (480 μL, 3.4 mmol) and mercury(II) chloride (192 mg, 0.7mmol) were added to a solution of Cbz-protected pseudothiourea III (280mg, 0.7 mmol) and 1,3-xylylene diamine (IVd, 50 mg, 0.3 mmol) in DMF (30mL). The reaction mixture was stirred at rt for 48 h and worked up thesame as in the procedure for Ib and followed by treatment of the crudeintermediate Vd with 30% HBr in acetic acid as previously described. Theresulting crude product (yellow solid) was crystallized from methanoland further purified by preparative HPLC on a symmetry C8 column [7μ,200×40 mm; flow rate=40 mL/min; mobile phase: MeCN/water (containing0.1% TFA); gradient:concentration of MeCN 5% for 0-2 min interval, thenincreased from 5% -20% MeCN from 2-10 min, 20% -60% MeCN from 10-30 min,60% -100% MeCN from 30-33 min and concentration decreased from 100% -5%MeCN from 33-35 min]. Product isolation and further treatment with HBras previously described gave the product Id as pale yellow solid: 31.2mg (12.1% yield from III); ¹H NMR (360 MHz, DMSO-d₆,) δ4.60 (d, J=5.2Hz, 4H), 7.40-7.42 (m, 7 h), 9.03 s, 4H), 9.61 (s, 2H) and 10.59 (s,2H); API MS m/z=561 [C₂₀H₂₂Cl₂N₁₄O ₂+H]⁺; HPLC (Method E) 97.3%, t_(r)=5.5 min.

EXAMPLE 7 1,5-Bis[(3,5-diamino-6-chloropyrazinoyl)guanidino]pentaneDihydrobromide (Ie)

Compound Ie was prepared following the same procedure described for Ib.The Cbz-protected pseudothiourea III (280 mg, 0.7 mmol) and1,5-diaminopentane (IVe, 37 mg, 0.35 mmol) were reacted in the presenceof triethylarnine (480 μL, 3.4 mmol) and mercury(GI) chloride (192 mg,0.7 mmol). The reaction mixture was stirred at rt for 24 h and worked upthe same as in the procedure for Ib and the resulting crude intermediateVe was treated with 30% HBr in acetic acid for 24 h as previouslydescribed. The reaction mixture was poured into ethyl ether (200 mL),the precipitate was collected by filtration, washed with ether, THF andthen crystallized twice from 12% HBr to give crude Ie (117 mg, 87%purity, 47% yield from III) as a yellow solid. A portion of thismaterial (78 mg) was crystallized again from 12% HBr to give Ie as apale yellow solid: 32 mg (12.8% yield from III);¹H NMR (360 MHz,DMSO-d_(6,)) δ1.39 (m, 2H), 1.61 (m, 4H), 3.31 (m, 4H), 7.44 (br s, 4H),8.72 (br 2H), 8.90 (br s, 2H), 9.20 (s, 2H) and 10.49 (s,2H); ¹³C NMR(90MHz, DMSO-d₆) δ23.1,27.3, 40.8,109.0, 119.7,153.1, 154.2,155.9 and165.2; API MS m/z=527 [C₁₇H₂₄Cl₂N₁₄O₂+H]³⁰; HPLC (Method E) 95.3%, t_(r)32 5.72 min.

EXAMPLE 8 1,5-Bis[(3,5-diamino-6-chloropyrazinoyl) ganidino]pentaneDihydrochloride (Ie 2 HCl)

The combined mother liquors from the crystallization of Ie were treatedwith powder KOH until the solution reached pH=11. The precipitate thatformed was collected by vacuum filtration, washed with water, andrecrystallized twice from 10% aqueous HCl to give Ie 2HCl as a paleyellow solid:27.3 mg (13% yield from III);¹H NMR (360 MHz, DMSO-d₆)δ1.40 (m, 2H), 1.61 (m, 4H), 3.34 (m, 4H), 7.41 (br s, 4H), 8.80 (br s,2H), 8.93 (br s, 2H), 9.29 (s, 2H) and 10.52 (s, 2H); ¹³C (90 MHzDMSO-d₆) δ23.1, 27.3, 40.8, 109.0, 119.7, 153.2, 154.2, 155.9 and 165.2;API MS m/z=527 [Cl₇H₂₄C1₂N₁₄0₂+H]³⁰; HPLC (Method E) 95.2%, t_(r)=5.

EXAMPLE 9 1,4-Bis [(3,5-diamino-6-chloropyrazinoyl)guanidino]xylyleneDihydrobromide (If)

Compound If was prepared following the same procedure described for Ib.The Cbz-protected pseudothiourea III (280 mg, 0.7 mmol) and1,4-xylylenediamine (IVf, 50 mg, 0.30 mmol) were reacted in the presenceof triethylamine (480 μL, 3.4 mmol) and mercury(II) chloride (192 mg,0.7 mmol). The reaction mixture was stirred at rt for 4 d and then itwas filtered through silica gel and concentrated under reduced pressure.The residue was suspended in anhydrous DMF (10 mL) and treated withtetrabutylammonium borohydride (50 mg, 0.17 mmol) in DMF (1 mL) andstirred for 15 min at rt to get rid of residual mercury(II) chloride.The reaction mixture was filtered through silica gel and concentrated togive a residue (Vf) that was treated with 30% HBr in acetic acid (20 mL)for 7 d at rt and 1 d at 45 ° C. The reaction mixture was poured intoether (200 mL) and the solid that precipitated was collected byfiltration, washed with ether, THF and crystallized twice from methanolto give a pale yellow solid: 74 mg (31% yield from III); ¹ H NMR (360MHz, DMSO-d₆) δ4.60 (d, J=4.3 Hz, 4H), 7.45 (s, 4H), 8.91 (br s, 2H),8.99 (br s, 2H 2H) and 10.56 (s, 2H); ¹³C NMR(90 MHz, DMSO-d₆) δ44.1,109.1, 119.7, 128.1, 135.4, 153.3, 154.3, 156.0 and 165.3; API MSm/z=561 [C₂₀H₂₂Cl₂N₁₄O₂+H]³⁰; HPLC (Method E) 95.7%, t_(r) =6.31 min.

EXAMPLE 101,8-Bis[(3,5-diamino-6-chloropyrazinoyl)guanidino]-3,6-dioxa-octaneDihydrochloride (Ig)

A solution of 2,2′-(ethylenedioxy)bis(ethylamine) (IVg, 45 mg, 0.3 mmol)in dry DMF (100 μL was added to III (240 mg, 0.7 mmol) in dry DMF (30mL), followed by addition of triethylamine (480 μL, 3.4 mmol) in DMF (1mL) and mercury(I) chloride (165 mg, 0.6 mmol) in DMF (600 μL). Thereaction mixture was stirred 16 h at room temperature, then additionalIII (20 mg) was added and reaction mixture was stirred an additional 8 hat 40 ° C. The reaction mixture was cooled to room temperature andtreated with tetrabutylammonium borohydride (50 mg, 0.17 mmol) in DMF (1mL) with stirring for 15 min at room temperature. The reaction mixturewas filtered through silica gel, concentrated under reduced pressure togive a solid residue. This was dissolved in 30% HBr in acetic acid (20mL) and stirred for 8 h at 40 ° C, then poured into ether (200 mL). Theresulting precipitate was collected by filtration and washed with ether.The solid was dissolved in water (25 mL), the solution filtered, and thefiltrate concentrated under reduced pressure. The resulting residue wasdissolved again in minimal 10% HBr and powdered NaOH is added to pH=11.A precipitate formed and was collected by filtration, washed with waterand dried to give the free base (98 mg, 56% yield). A portion of thismaterial (58 mg) was dissolved in 10% HCl and then concentrated underreduced pressure. The residue is washed with ether and dried to give Igas a pale yellow solid: 64 mg (32% yield from III);¹H NMR (360 MHz,DMSO-d₆) 8 3.56 (br s, 4H), 3.63 (br s, 8H), 7.4 (br s, 4H), 9.09 (br s,4H), 9.52 (br s, 2H) and 10.70 (s, 2H); ¹³C NMR ( MHz, DMSO-d₆) δ41.1,67.7, 69.5, 108.9, 119.7, 153.3, 154.2, 155.8 and 165.3; API MS m/z =573[C₁₈H₂₆Cl₂N₁₄O₄ +H]³⁰; LC (Method C) 97.6%, t_(r)=4.2

EXAMPLE 11 Potency of Dimeric Compounds

Two pharmacologic assays were used to determine the relative potency ofthe dimers described herein. The first assay examined the expression ofthe subunits α, β, and γ of recombinant apical membrane epithelial Na⁺channel (or “rENaC”) in Xenopus oocyte, as follows: cRNAs for all threeENaC subunits were injected into oocytes via conventional microinjectiontechniques. After two to three days, two electrode voltage clampprotocols were used to measure ENaC-mediated Na⁺currents. Test compoundswere assayed using cumulative drug addition protocols known in the art.Single oocytes were used for single compounds. Compounds tested werethen compared to dose-effect relationships for amiloride and benzamil inthe same batch of injected eggs.

In the second potency assay, airway epithelial monolayers mounted werein Using chambers:The principal assay consisted of tests of lumenal druginhibition of airway epithelial Na⁺ currents. Cells obtained fromfreshly excised human or dog airways were seeded onto SNAP-well Inserts(CoStar), cultured under air-liquid (ALI) conditions in hormonallydefined media. The cells were assayed for Na⁺ transport activity whilebathed in Krebs bicarbonate Ringer (KBR) in the Ussing chambers undervoltage clamp conditions. All test drug additions were to the mucosalbath with half-log dose addition protocols (10⁻¹¹M-10⁻⁵ M). All drugswere made in standard stocks of 10⁻² M drug in DMSO. Eight preparationswere typically run in parallel; two preparations/run were routinely usedto assay amiloride and benzamil. After the maximal concentration (10⁻⁴M) was administered, the lumenal bath was exchanged three times withfresh KBR solution, which was defined as the “wash-out” effect. All datafrom the voltage clamps were collected via a computer interface andanalyzed off-line.

Dose-effect relationships for all compounds were considered and analyzedby the Prism 3.0 program. The IC₅₀, maximal effective concentrations,and percent washout were calculated and compared to those of amilorideand benzamil as reference compounds.

EXAMPLE 12 Absorption Assays

Compounds useful in the present invention preferably have thecharacteristics of high potency and non- or decreased absorbability intomucosal surfaces. Two pharmacologic assays were employed to test theabsorption of compounds illustrated in TABLE 1.

The first assay is referred to an assay of reversibility. Empirically,the percent of wash-out correlates with cellular uptake. Therelationship is complex because reversibility is also a function ofpotency. However, reversibility is a quick and simple screen. An exampleof the information obtained by such as assay is shown in FIG. 1. Thecompounds that “reverse” best in this assay referenced to benzamil werealso the least absorbed in the confocal assay, as described below.

The second assay utilizes confocal microscopy of amiloride congeneruptake: Virtually all amiloride-like molecules fluoresce in UV range.This property of these molecules was used to directly measure cellularuptake, using a x-z confocal microscope (Leica). As an example of theresults obtained by this assay are shown in FIG. 2. In the experimentshown in FIG. 2, equimolar concentrations of amiloride and compounds ofrapid (benzamil) and very rapid uptake (phenamil) were placed on theapical surface of airway cultures on the stage of the confocalmicroscope. Serial x-z images were obtained with time and the magnitudeof fluorescence accumulating in the cell compartment quantitated andplotted. The assay was subsequently optimized to test for compounds thatabsorbed into cells less rapidly than amiloride. Two compounds from thesynthesis series described above (CF-509 and CF-519) appear to fulfilthis criterion. Compounds that were equipotent or greater with amiloridewere tested for wash-out as described above. However, because wash-outmay reflect both potency and cell uptake, the rate of accumulation offluorescence (indexed to the specific fluorescence/emission spectrum ofeach compound) in the cell compartment as a function of time was alsoroutinely measured. The relative cellular uptake of each test compoundwas then compared to the reference compounds (amiloride, benzamil) asfor potency assays.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A pharmaceutical formulation in apharmaceutically acceptable carrier solution, comprising the compound ofthe formula P₁-L-P₂, wherein: P₁ is a pyrazinoylguanidine sodium channelblocker; L is a linking group; and P₂ is selected from the groupconsisting of pyrazinoylguanidine sodium channel blockers and P2Y₂receptor agonists; or a pharmaceutically acceptable salt thereof.
 2. Thepharmaceutical formulation according to claim 1, wherein P₂ is apyrazinoylguanidine sodium channel blocker.
 3. The pharmaceuticalformulation according to claim 1, wherein: P₁ and P₂ are eachindependently selected from the group consisting of compounds of theformula:

wherein: X is selected from the group consisting of chioro, bromo, iodo,loweralkyl, lower-cycloalkyl having from 3 to 7 carbons, phenyl,chlorophenyl, bromophenyl, Z-thio and Z-sulfonyl wherein Z is selectedfrom the group consisting of loweralkyl, oxyalkyl, andphenyl-loweralkyl; Y is selected from the group consisting of hydroxyl,mercapto, loweralkyloxy, loweralkylthio, chloro, loweralkyl,lowercycloalkyl having from 3 to 6 carbons, phenyl, and amino having thestructure:

wherein: R is selected from the group consisting of hydrogen, amino,amidino, lower-cycloalkyl having 3 to 6 carbon atoms, loweralkyl,hydroxyloweralkyl, halo-loweralkyl, lower-(cycloalkylalkyl) having 3 to6 carbons in the ring, phenyl-loweralkyl, lower-(alkylaminoalkyl),lower-alkenyl, phenyl, halophenyl, and lower-alkylphenyl; R₁ is selectedfrom the group consisting of hydrogen, loweralkyl, loweralkenyl, andadditionally; R and R₁ can be joined to form a lower alkylene; R₂ isselected from the group consisting of hydrogen and loweralkyl; R₃ and R₄are independently selected from the group consisting of hydrogen,loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-(azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl; and L is selected from thegroup consisting of loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl.
 4. The pharmaceuticalformulation according to claim 1, wherein P₂ is a nucleotide ordinucleotide P₂Y₂ receptor agonist.
 5. The pharmaceutical formulationaccording to claim 1, wherein P₂ is a dinucleotide having the formula:

wherein: A and B are each independently a purine or pyrimidine base; R₁and R₂ are each independently selected from the group consisting of H orOH; X is OH, SH, O or S; and n is from 1 to 6; with said dinucleotidecovalently joined to L by covalent attachment to A or B or covalentattachment to the ribose or deoxyribose ring to which A and B arejoined.
 6. The pharmaceutical formulation according to claim 1, whereinthe compound has the structure:


7. The pharmaceutical formulation according to claim 1, wherein thecompound has the structure:


8. The pharmaceutical formulation according to claim 1, wherein P₁ isselected from the group consisting of amiloride, benzamil and phenamil.9. The pharmaceutical formulation according to claim 1, wherein thepharmaceutical formulation is useful for treating a mucosal surface in asubject in need thereof.
 10. The pharmaceutical formulation according toclaim 9, wherein the mucosal surface is an airway mucosal surface. 11.The pharmaceutical formulation according to claim 9, wherein the mucosalsurface is a gastrointestinal mucosal surface.
 12. The pharmaceuticalformulation according to claim 9, wherein the subject is a human oranimal subject.
 13. The pharmaceutical formulation according to claim 9,further comprising at least one other compound useful in the treatmentof a mucosal surface in a subject in need thereof.
 14. Thepharmaceutical formulation according to claim 1, wherein thepharmaceutical formulation is suitable for administration to a subjectin thereof.
 15. The pharmaceutical formulation according to claim 14,wherein the pharmaceutical formulation is suitable for topicaladministration.
 16. The pharmaceutical formulation according to claim14, wherein the pharmaceutical formulation is suitable for oraladministration.
 17. The pharmaceutical formulation according to claim14, wherein the pharmaceutical formulation is suitable for rectaladministration.
 18. The pharmaceutical formulation according to claim14, wherein the pharmaceutical formulation is suitable for inhalationaladministration.
 19. The pharmaceutical formulation according to claim14, wherein the pharmaceutical formulation is suitable for transdermaladministration.
 20. The pharmaceutical formulation according to claim14, wherein the pharmaceutical formulation is suitable for parenteraladministration.